WO2011060634A1

WO2011060634A1 - 23-hydroxy-betulinic acid derivatives, preparation methods and uses thereof

Info

Publication number: WO2011060634A1
Application number: PCT/CN2010/072740
Authority: WO
Inventors: 张南; 钟荣
Original assignee: 苏州麦迪仙医药科技有限公司
Priority date: 2009-11-23
Filing date: 2010-05-13
Publication date: 2011-05-26
Also published as: CN101704872A; US20120302530A1; CN101704872B

Abstract

23-hydroxy-betulinic acid derivatives, including 3-oxo-23-hydroxy-betulinic acid, 3-oxo-23-hydroxy-dihydrobetulinic acid and 23-hydroxy-dihydrobetulinic acid, their preparation methods and medical uses as antitumor and anti-HIV agents are disclosed.

Description

23-羟基白桦酸衍生物、其制备方法及应用技术领域 23-hydroxy betulinic acid derivative, preparation method and application thereof

本发明涉及具有抗肿瘤和抗艾滋病作用的 23-羟基白桦酸衍生物、其制备方法及应用。背景技术 The present invention relates to a 23-hydroxy betulinic acid derivative having antitumor and anti-AIDS effects, a preparation method and application thereof. Background technique

白桦酸，又名桦木酸，是五环三萜类化合物，英文名称为 Betiilinic acid , 化学名为 3 p-hydroxy-lup-20(29)-ene-28-oic Birch acid, also known as betulinic acid, is a pentacyclic triterpenoid, English name is Betiilinic acid, chemical name is 3 p-hydroxy-lup-20(29)-ene-28-oic

acid , 分子式为 C₃。H₄₈0₃，分子量为 456.71。白桦酸有几种生物来源，如从蒲桃树叶、白桦树皮乙醇萃取物进行重结晶提取；也可以用白桦脂醇为原料通过化学合成得到。可用作调香剂。白桦酸是一种无色结晶，熔点 316〜318 °C，旋光度 [a】_D +8°，能溶于***、乙醇、丙酮、氯仿、吡啶。白桦酸的性质和作用可参见下面三篇文献：周映霞，王振国，马莉，阎浩林，白桦酯酸及其衍生物的研究概况，中国药学杂志， 2005年 06期；徐萍，周金培，徐进宜，吴晓明，白桦酸类化合物抗肿瘤活性的研究进展，中国药师， 2006年 02期；李丹，周金培，吴晓明，白桦酸及其衍生物的研究进展，药学进展， 2004年 03期，白桦酸具有抗肿瘤、抗艾滋病病毒、抗炎和体外抗疟病作用等多种生物活性，并对肿瘤细胞具有一定的选择性细胞毒性及其较好的治疗指数，甚至达到 500mg/kg时，也没有明显的毒性反应。九十年代中期经研究发现白桦脂酸可以选择性地杀死人类黑色素瘤细胞而不杀伤健康细胞；另外近期的研究表明白桦酸对脑瘤、神经外胚层瘤、白血病等恶性肿瘤细胞也有抑制作用。白桦酸抗肿瘤作用机制认为是降低肿瘤细胞线粒体膜电位，引起细胞内线粒体通透性改变，最后导致细胞凋亡，另外与其上调肿瘤细胞膜 p53蛋白的表达，改变肿瘤细胞膜 Ca²⁺、 Mg²⁺-ATP酶活性有关，具体参见两篇文献，叶银英，何道伟，叶文才，张庆文，赵守训 . 23-羟基桦木酸体外和体内抗黑色素瘤作用的研究 [J】中国肿瘤临床与康复， 2000年 01期；程燕，叶文才，石俊敏，姚新生，栗原博. 23-羟基白桦酸诱导 LoVo细胞凋亡与线粒体膜电位的变化 [J] . 中国药理学通报， 2007年 03期。 Acid , the molecular formula is C ₃ . H ₄₈ 0 ₃ , molecular weight is 456.71. There are several biological sources of betulinic acid, such as recrystallization from the extract of rose apple leaves and bark of bark; it can also be obtained by chemical synthesis using betulin as a raw material. Can be used as a flavoring agent. Birch acid is a colorless crystal with a melting point of 316~318 °C, optical rotation [a] _D +8 °, soluble in ether, ethanol, acetone, chloroform, pyridine. The properties and effects of betulinic acid can be found in the following three documents: Zhou Yingxia, Wang Zhenguo, Ma Li, Qi Haolin, Research on the study of betulinic acid and its derivatives, Chinese Journal of Pharmacy, 2005, 06; Xu Ping, Zhou Jinpei, Xu Jinyi, Wu Xiaoming, Advances in antitumor activity of betulinic acid compounds, Chinese pharmacist, 2006, 02; Li Dan, Zhou Jinpei, Wu Xiaoming, Research progress of betulinic acid and its derivatives, Progress in Pharmaceutical Sciences, 2004, 03, Birch acid has anti-tumor, Anti-HIV, anti-inflammatory and anti-malarial effects in vitro, and have certain selective cytotoxicity and good therapeutic index for tumor cells. Even when it reaches 500mg/kg, there is no obvious toxicity. . In the mid-1990s, it was found that betulinic acid can selectively kill human melanoma cells without killing healthy cells. In addition, recent studies have shown that betulinic acid can also inhibit malignant tumor cells such as brain tumors, neuroectodermal tumors and leukemia. . The anti-tumor mechanism of betulinic acid is thought to reduce the mitochondrial membrane potential of tumor cells, cause changes in intracellular mitochondrial permeability, and finally lead to apoptosis. In addition, it up-regulates the expression of p53 protein in tumor cell membrane and changes the membranes of Ca ²⁺ and Mg ^{2+ .} -ATPase activity, see two articles, Ye Yinying, He Daowei, Ye Wencai, Zhang Qingwen, Zhao Shouxun. Study on the anti-melanoma effect of 23-hydroxy betulinic acid in vitro and in vivo[J] Chinese Journal of Clinical Oncology and Rehabilitation, 2000-01 Cheng Yan, Ye Wencai, Shi Junmin, Yao Xin Sheng, Li Yuanbo. Changes of Apoptosis and Mitochondrial Membrane Potential in LoVo Cells Induced by 23-Hydroxy-Betulinic Acid[J]. Chinese Journal of Pharmacology, 2007, 03.

传统中药白头翁，具清热解毒、活血化瘀功效，中医临床主要用于结肠癌、直肠癌等肠道肿瘤及子***、脑垂体瘤、甲状腺瘤、肺癌的治疗，具体参见文献 Ye w c， Zhang Q W, Hsiao W L , et al . New lupane glycosides from pulsatila chinensis [J] . P / anta Medwa , 2002， 68： 183-6。 23-羟基白桦酸是从白头翁中发现的三萜皂苷元，其含量高达 2.5 %，参见文献叶银英，何道伟，叶文才，等. 23—羟基桦木酸体外和体内抗黑色素肿瘤作用的研究 [J】. 中国肿瘤临床与康复， 2000 7(1)： 5 一 7。近年来的研究表明 23-羟基白桦酸在体内、外均能够选择性抑制小鼠黑色素瘤 B16细胞的增殖，其作用机制主要是通过诱导 B16细胞凋亡，参见两篇文献 Ye Y Y， He D W, Ye W C , et al . The study of 23-hydroxybetulinic Traditional Chinese medicine Pulsatilla chinensis has the functions of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis. Traditional Chinese medicine is mainly used for the treatment of colon cancer, rectal cancer and other intestinal tumors and cervical cancer, pituitary tumor, thyroid tumor and lung cancer. For details, see Ye wc, Zhang QW, Hsiao WL, et al. New lupane glycosides from pulsatila chinensis [J] . P / anta Medwa, 2002, 68: 183-6. 23-hydroxy betulinic acid is a triterpenoid saponin found in Pulsatillae, its content is as high as 2.5%, see literature Ye Yinying, He Daowei, Ye Wencai, et al. Study on the anti-melanoma effect of 23-hydroxy betulinic acid in vitro and in vivo [J] Chinese Journal of Clinical Oncology and Rehabilitation, 2000 7(1): 5-7. Recent studies have shown that 23-hydroxy-cedaric acid can selectively inhibit the proliferation of mouse melanoma B16 cells in vitro and in vivo, and its mechanism is mainly through the induction of apoptosis of B16 cells. See two articles Ye YY, He DW, Ye WC , et al . The study of 23-hydroxybetulinic

Acid against melanoma in vivo and in vitro [J] . Chin J Clin Acid against melanoma in vivo and in vitro [J] . Chin J Clin

Oncol Oncol

Rehab. 2000, 7(1)： 5—7 ; Wu W K , Ho J C， Cheung F W ， et Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line [J] . Eur J Pharmacol. 2004 , 498(1—3) : 71— 8。 Rehab. 2000, 7(1): 5-7; Wu WK, Ho JC, Cheung FW, et Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line [J] . Eur J Pharmacol. 2004 , 498 (1-3) ) : 71-8.

综上，白桦酸和 23-羟基白桦酸具有抗肿瘤作用，因此可用作抗肿瘤的治疗药。目前，抗肿瘤药物应用于人体时，不同个体的药效作用是有很大差异的，抗肿瘤药物的分子作用机理现在也没有完全研究透彻，因此不同个体只能从不同的抗肿瘤药物中选择对自己药效作用大，副作用小的药物，则对开发更多的抗肿瘤药物或者具有抗肿瘤活性的化合物是很有需求的。基于白桦酸和 23-羟基白桦酸具有的抗肿瘤作用，人们会期待从白桦酸或 23-羟基白桦酸的衍生物中发现其他具有生物活性的化合物，这些化合物可以应用于抗肿瘤的治疗药，或者具有其他作用，如抗艾滋病。为达到上述目的，发明人对白桦酸或 23-羟基白桦酸的很多衍生物进行了研究，结果发现了三种 23-羟基白桦酸衍生物是具有抗肿瘤和抗艾滋病活性的，从而完成了本发明。发明内容 In conclusion, betulinic acid and 23-hydroxycetanoic acid have antitumor effects and are therefore useful as antitumor therapeutic agents. At present, when anti-tumor drugs are applied to human body, the pharmacodynamic effects of different individuals are very different. The molecular mechanism of anti-tumor drugs is not fully studied now, so different individuals can only choose from different anti-tumor drugs. Drugs with a large effect on their own efficacy and small side effects are highly desirable for the development of more antitumor drugs or compounds with antitumor activity. Based on the anti-tumor effect of betulinic acid and 23-hydroxy-cedaric acid, it is expected that other biologically active compounds will be found in derivatives of betulinic acid or 23-hydroxy-cedaric acid, and these compounds can be applied to anti-tumor therapeutic drugs. Or have other effects, such as fighting AIDS. In order to achieve the above object, the inventors studied many derivatives of betulinic acid or 23-hydroxycetanoic acid, and found three kinds of 23-hydroxy betulinic acid derivatives. The present invention has antitumor and anti-AIDS activities, thereby completing the present invention. Summary of the invention

本发明的第一个目的是提供具有抗肿瘤和抗艾滋病作用的新化合物，该新化合物是属于 23-羟基白桦酸的衍生物；本发明的第二个目的是提供新化合物的制备方法；本发明的第三个目的是说明新化合物的应用。 A first object of the present invention is to provide a novel compound having antitumor and anti-AIDS effects, which is a derivative belonging to 23-hydroxycetanoic acid; a second object of the present invention is to provide a method for preparing a novel compound; A third object of the invention is to illustrate the use of new compounds.

本发明提供了三种新的 23-羟基白桦酸衍生物，化学名称为 3-羧基 -23-羟基白桦酸、 3-羧基 -23-羟基二氢白桦酸、 23-羟基二氢白桦酸，化学式依次为 The present invention provides three new 23-hydroxy betulinic acid derivatives, the chemical name is 3-carboxy-23-hydroxy betulinic acid, 3-carboxy-23-hydroxydihydroglycylic acid, 23-hydroxydihydrocetanoic acid, chemical formula as followed

3-羧基 -23-羟基白桦酸，英文名称为 3-carbonyl-23-hydroxy betulinic acid, 其分子式为 C₃。H₄₆0₄，分子量为 470.68，其制备方法如下： 3-carboxy-23-hydroxy betulinic acid, English name is 3-carbonyl-23-hydroxy betulinic acid, and its molecular formula is C ₃ . H ₄₆ 0 ₄ , molecular weight is 470.68, and its preparation method is as follows:

( 1 ) 将 500mg 23-羟基白桦酸溶解于 1ml吡啶中，于零下 5度下搅拌均匀，缓慢滴加醋酸酑 0.15ml，而后反应于零下 5度搅拌反应约 24小时后处理反应。于该温度下缓慢滴加氯化铵饱和溶液，直至无气泡产生，乙酸乙酯萃取三次后合并有机层，饱和氯化钠水溶液洗有机层两次，再用无水硫酸钠干燥，旋干有机溶剂乙酸乙酯后上柱分离，除回收部分原料外，分别得到化合物 3， 23-二乙酰基 -23-羟基白桦酸 (定义为 zhangiiaii-1 )， 23-二乙酰基 -23-羟基白桦酸（定义为 zhangnan-2 ) 和 3-二乙酰基 -23-羟基白桦酸（定义为 zhangnan-6 )，化合物 zhangnaii-l、zhaiigiiaii-2、 zhangnan-6分另 [J占总产物的 30% , 60% 和 10% , 该反应骤的总收率为 65% , 反应式如下： (1) 500 mg of 23-hydroxy betulinic acid was dissolved in 1 ml of pyridine, stirred at minus 5 degrees, and 0.15 ml of cesium acetate was slowly added dropwise, and then the reaction was stirred at minus 5 degrees for about 24 hours to treat the reaction. At this temperature, a saturated solution of ammonium chloride was slowly added dropwise until no bubbles were formed. The organic layer was extracted three times with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. The solvent is ethyl acetate and then separated on the column. In addition to recovering some of the starting materials, the compound 3, 23-diacetyl-23-hydroxybirthic acid (defined as zhangiiaii-1), 23-diacetyl-23-hydroxyglycine (defined as zhangnan-2) and 3 were obtained, respectively. -diacetyl-23-hydroxycetanoic acid (defined as zhangnan-6), compound zhangnaii-l, zhaiigiiaii-2, zhangnan-6, respectively [J accounted for 30%, 60% and 10% of the total product, the reaction The total yield is 65%, and the reaction formula is as follows:

( 2 )将 lOOmg 化合物 zhangnan-2 溶解分散于 30ml二氯甲烷中，于冰浴下加入氧化剂二环己基碳二亚胺 58mg后，于室温搅拌反应 4 小时后处理，旋干溶剂二氯甲烷后用乙酸乙酯溶解后用粗硅胶进行柱分离，得到化合物 3-羧基 -23-二乙酰基 -23-羟基白桦酸（定义为 zh ； (2) Dissolve and dissolve 100 mg of the compound zhangnan-2 in 30 ml of dichloromethane, add 58 mg of the oxidant dicyclohexylcarbodiimide in an ice bath, stir the reaction at room temperature for 4 hours, and then spin dry the solvent in dichloromethane. After dissolving with ethyl acetate, the column was separated with crude silica gel to obtain the compound 3-carboxy-23-diacetyl-23-hydroxybutteric acid (defined as zh;

( 3 ) 取 50mg 化合物 zhangnan-4 分散溶解于 30ml甲醇中，加入 (3) Take 50mg of compound zhangnan-4, disperse and dissolve in 30ml of methanol, add

5ml碳酸钾的饱和溶液后，于室温搅拌反应 2小时后处理反应，乙酸乙酯萃取三次后合并有机层，饱和氯化钠水溶液洗有机层两次，再用无水硫酸钠干燥，旋干有机溶剂乙酸乙酯后上柱分离，得化合物 3- 羧基 -23-羟基白桦酸（定义为 zhangnaii-5 ) 40mg , 该反应步骤的收率为 90% , 反应式如下： After 5 ml of a saturated solution of potassium carbonate, the reaction mixture was stirred at room temperature for 2 hours, and then the mixture was combined, and the organic layer was combined, and the organic layer was washed twice with saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. The solvent was subjected to ethyl acetate and then separated on a column to obtain 40-mg of the compound 3-carboxy- 23-hydroxy betulinic acid (defined as zhangnaii-5). The yield of the reaction step was 90%, and the reaction formula was as follows:

化合物 zhangnan-5

Compound zhangnan-5

H-NMR(400 MHz, CDC13 ) 6： 0.963, 0.969, 0.992, 1.015， 1.690(各 3H， s， 5XCH3), 2.99(1H， m， H-19), 4.71(1H, d, J=1.6 Hz, 29-H), 4. 61(1H, d, J=1.6 Hz, 29-H)。 H-NMR (400 MHz, CDC13) 6: 0.963, 0.969, 0.992, 1.015, 1.690 (3H, s, 5XCH3), 2.99 (1H, m, H-19), 4.71 (1H, d, J = 1.6 Hz , 29-H), 4. 61 (1H, d, J = 1.6 Hz, 29-H).

C-NMR(400 MHz, CDC13) δ: 213.95, 182.21, 150.31, 109.79, 56.36, 53.30, 49.20, 48.17, 46.93, 44.58, 42.56, 40.38, 40.34, 38.41, 37.43, 37.04, 36.79, 33.08, 32.13, 30.56, 29.60, 25.46, 22.18, 21.51, 19.34, 15.89, 14.52, 13.54。 C-NMR (400 MHz, CDC13) δ: 213.95, 182.21, 150.31, 109.79, 56.36, 53.30, 49.20, 48.17, 46.93, 44.58, 42.56, 40.38, 40.34, 38.41, 37.43, 37.04, 36.79, 33.08, 32.13, 30.56 , 29.60, 25.46, 22.18, 21.51, 19.34, 15.89, 14.52, 13.54.

MS(ESI)： m/ z 440 [M-20 MS (ESI): m/z 440 [M-20

3-羧基 -23-羟基二氢白桦酸，英文名称为 3-carbonyl-23-hydroxy Dihydrobetulinic acid, 其分子式为 C₃。H₄₈0₄，分子量为 472.7，其制备方法如下： 3-carboxy-23-hydroxydihydroglycine, English name is 3-carbonyl-23-hydroxy Dihydrobetulinic acid, and its molecular formula is C ₃ . H ₄₈ 0 ₄ , molecular weight 472.7, the preparation method is as follows:

取 30mg 化合物 zhangnan-5 分散溶解于 30ml 甲醇中，加入约 10mg还原剂钯碳后密封反应体系，抽真空，充入氢气，并于氢气流中室温搅拌反应 10 小时后结束反应，滤除钯碳后旋干溶剂，得到产物 3-羧基 -23-羟基二氢白桦酸（定义为 zhangnan-7) 25mg, 该反应的收率 30 mg of compound zhangnan-5 was dispersed and dissolved in 30 ml of methanol, about 10 mg of reducing agent palladium carbon was added, and the reaction system was sealed, vacuumed, charged with hydrogen, and stirred at room temperature for 10 hours in a hydrogen stream to terminate the reaction, and the palladium carbon was filtered off. After drying the solvent to give the product 3-carboxy-23-hydroxydihydrobelovic acid (defined as zhangnan-7) 25 mg, the yield of the reaction

化合物 zhangnan-7

Compound zhangnan-7

H-NMR(400 MHz, CDC13 ) δ: 0.755(3H, d, 3=1.2 Hz), 0.963(3H， d, 3=1.2 Hz), 0.894， 0.936， 1.048， 1.301(各 3H， s, 4XCH3), 2.243(4H, m)。 H-NMR (400 MHz, CDC13) δ: 0.755 (3H, d, 3 = 1.2 Hz), 0.963 (3H, d, 3 = 1.2 Hz), 0.894, 0.936, 1.048, 1.301 (3H, s, 4XCH3 each) , 2.243(4H, m).

C-NMR(400 MHz, CDC13) δ: 212.69, 177.79, 56.29, 52.98, 48.52, 47.65, 44.19, 44.12, 42.71,37.87, 37.33, 37.22, 36.78, 33.16, 32.12, 29.82, 29.59, 27.02, 23.45, 22.93, 22.19, 21.59, 16.12, 14.99, 14.58, 13.63, 12.04。 C-NMR (400 MHz, CDC13) δ: 212.69, 177.79, 56.29, 52.98, 48.52, 47.65, 44.19, 44.12, 42.71, 37.87, 37.33, 37.22, 36.78, 33.16, 32.12, 29.82, 29.59, 27.02, 23.45, 22.93 , 22.19, 21.59, 16.12, 14.99, 14.58, 13.63, 12.04.

MS(ESI)： ml z 442 [M-20H] 。 MS (ESI): ml z 442 [M-20H].

23- 羟基二氢白桦酸，英文名称为 23-hydroxy Dihydrobetulinicacid, 其分子式为 C₃。H₄。0₄，分子量为 474.72，其制备方法如下： 23-Hydroxydihydrobutteric acid, English name is 23-hydroxy Dihydrobetulinicacid, and its molecular formula is C ₃ . H ₄ . 0 ₄ , the molecular weight is 474.72, and the preparation method is as follows:

取 30mg化合物 23-羟基白桦酸分散溶解于 30ml 甲醇中，加入约 10mg还原剂钯碳后密封反应体系，抽真空，充入氢气，并于氢气流中室温搅拌反应 10 小时后结束反应，滤除钯碳后，旋干溶剂得到产物 23-羟基二氢白桦酸（定义为 zhangnan-8)20mg，该反应的收率为 70%，反应式如下： 30 mg of compound 23-hydroxy betulinic acid was dispersed and dissolved in 30 ml of methanol, about 10 mg of reducing agent palladium carbon was added, and the reaction system was sealed, vacuumed, charged with hydrogen, and stirred at room temperature for 10 hours in a hydrogen stream to terminate the reaction, and filtered. After palladium on carbon, the solvent was evaporated to give the product 23-hydroxydihydrobelovic acid (defined as zhangnan-8) 20 mg, the yield of the reaction was 70%, and the reaction formula was as follows:

化合物 zhangnan-8 Compound zhangnan-8

H-NMR(400 MHz, CDC13 ) δ: 0.727(3Η, d, J=6.4 Hz ， CH3)， 0.816(3H, d, J=6.8 Hz ， CH3), 0.790, 0.853， 0.910， 1.289 (各 3H， s， 4XCH3), 2.243(4H， m)。 H-NMR (400 MHz, CDC13) δ: 0.727 (3 Η, d, J = 6.4 Hz, CH3), 0.816 (3H, d, J = 6.8 Hz, CH3), 0.790, 0.853, 0.910, 1.289 (each 3H, s, 4XCH3), 2.243(4H, m).

C-NMR(400 MHz, CD30D) δ: 70.58, 64.82, 56.33, 50.39, 48.98, 47.09, C-NMR (400 MHz, CD30D) δ: 70.58, 64.82, 56.33, 50.39, 48.98, 47.09,

44.37, 42.68, 42.49, 39.38, 38.76, 37.64, 36.90, 34.26, 29.96, 27.37 27.18, 23.62, 23.23, 21.17, 17.99, 16.81, 16.66, 15.22, 14.78, 12.89。 44.37, 42.68, 42.49, 39.38, 38.76, 37.64, 36.90, 34.26, 29.96, 27.37 27.18, 23.62, 23.23, 21.17, 17.99, 16.81, 16.66, 15.22, 14.78, 12.89.

MS(ESI)： m/ z 444 [M-20H] 。 MS (ESI): m/z 444 [M-20H].

三种新的 23-羟基白桦酸衍生物， 3-羧基 -23-羟基白桦酸、 3-羧基 -23- 羟基二氢白桦酸、 23-羟基二氢白桦酸，其制备方法可参考文献 Hashimoto F, Kashiwada Y, Cosentino LM, Chen CH, Garrett PE, Lee KH.， Anti-AIDS agents-XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorg Med Chem. 1997 Dec;5(12):2133-43。该三种新化合物能广泛抑制多种类型的人肿瘤细胞系生长，并进一步诱导肿瘤细胞凋亡。在人肿瘤异种移植模型研究中， 3-羧基 -23-羟基白桦酸被证明是一种有效的抗肿瘤剂，能强烈抑制人黑色素瘤、白血病、恶性神经胶质瘤、 ***癌、肺癌和结肠癌细胞在体内生长。 3-羧基 -23-羟基白桦酸具有被开发成为新的广谱抗肿瘤药物的巨大潜能。本发明还发现 3-羧基 -23-羟基白桦酸、 3-羧基 -23-羟基二氢白桦酸和 23-羟基二氢白桦酸还具有抗 HIV病毒作用，特别是 3-羧基 -23-羟基二氢白桦酸抗 HIV病毒作用最强并且强过临床上使用的抗艾滋病药物阳性对照组。因此说， 3-羧基 -23-羟基二氢白桦酸具有被开发成为新的抗艾滋病药物的巨大潜能。 Three new 23-hydroxy betulinic acid derivatives, 3-carboxy-23-hydroxy betulinic acid, 3-carboxy-23-hydroxydihydroglycine, 23-hydroxydihydroglycoic acid, the preparation method can be found in Hashimoto F , Kashiwada Y, Cosentino LM, Chen CH, Garrett PE, Lee KH., Anti-AIDS agents-XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorg Med Chem. 1997 Dec;5(12): 2133-43. These three new compounds can inhibit the growth of many types of human tumor cell lines and further induce tumor cell apoptosis. In the study of human tumor xenograft models, 3-carboxy-23-hydroxy-cedaric acid has been shown to be an effective anti-tumor agent that strongly inhibits human melanoma, leukemia, malignant glioma, prostate cancer, lung cancer and colon Cancer cells grow in the body. 3-Carboxy-23-hydroxycetanoic acid has great potential to be developed as a new broad-spectrum anti-tumor drug. The present invention also finds that 3-carboxy-23-hydroxycetanoic acid, 3-carboxy-23-hydroxydihydrocedaric acid and 23-hydroxydihydrocetanoic acid also have anti-HIV virus effects, especially 3-carboxy-23-hydroxyl Hydrogen cetulinic acid has the strongest anti-HIV effect and is stronger than the clinically used anti-AIDS drug positive control group. Therefore, 3-carboxy-23-hydroxydihydrocetanoic acid has great potential to be developed as a new anti-AIDS drug.

附图说明 DRAWINGS

附图 1为化合物 zhangnaii-5或对照组治疗皮下接种的人*** 癌（LnCAP ) 肿瘤细胞的肿瘤体积一移植后天数曲线图； Figure 1 is a graph showing the tumor volume of a human prostate cancer (LnCAP) tumor cell subcutaneously inoculated with a compound zhangnaii-5 or a control group;

附图 2 为化合物 zhangnaii-5 或对照组治疗皮下接种的人肺癌 ( NCI-H23 ) 肿瘤细胞的肿瘤体积一移植后天数曲线图； Figure 2 is a graph showing the tumor volume of a human lung cancer (NCI-H23) tumor cell subcutaneously inoculated with a compound zhangnaii-5 or a control group;

附图 3为化合物 zhangnaii-5或对照组治疗皮下接种的人结肠癌 ( HCT-116 ) 肿瘤细胞的肿瘤体积一移植后天数曲线图； Figure 3 is a graph showing the tumor volume-day days after transplantation of the compound zhangnaii-5 or the control group for treating subcutaneously inoculated human colon cancer (HCT-116) tumor cells;

附图 4为化合物 zhangnaii-5或对照组治疗皮下接种的人黑色素瘤（G-361 ) 肿瘤细胞的肿瘤体积一移植后天数曲线图； Figure 4 is a graph showing the tumor volume-day days after transplantation of the compound zhangnaii-5 or the control group for treating subcutaneously inoculated human melanoma (G-361) tumor cells;

附图 5为化合物 zhangnaii-5或对照组治疗皮下接种的人恶性神经胶质瘤（U-251 ) 肿瘤细胞的肿瘤体积一移植后天数曲线图。具体实施方式 Figure 5 is a graph showing the tumor volume-day days after transplantation of the compound zhangnaii-5 or the control group for human malignant glioma (U-251) tumor cells subcutaneously inoculated. detailed description

下面结合附图来进一步阐述本发明。 The invention is further illustrated below in conjunction with the drawings.

本发明涉及三种新的 23-羟基白桦酸衍生物，化合物 _Zh_{a n}g_{n an}-5、 zhangnan-7和 zhangnan-8，它们的化学名称、化学式、表征化学结构的各种参数以及制备方法已在发明内容部分公开，这里通过实验来说明三种新化合物的生物活性。 The present invention relates to three novel 23-hydroxy betulinic acid derivatives, compounds _Z h _an g _{n an} -5, zhangnan-7 and zhangnan-8, their chemical names, chemical formulas, various parameters for characterizing chemical structures, and preparation methods It has been disclosed in the Summary of the Invention, where the biological activities of the three new compounds are illustrated by experiments.

( 1 ) 化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8广泛抑制人体肿瘤细胞株，但对正常细胞株没有作用：（可参考文献 Carmichael J, DeGraff WG, Gazadar AF, Minna JD, Mitchell JB; Evaluation of a tetrazolium-based semiautomated colormeteric assay: assessment of c he mo sensitivity testing. Cancer Research, 1987, 47: 943-946。） (1) Compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 broadly inhibit human tumor cell lines, but have no effect on normal cell lines: (Refer to Carmichael J, DeGraff WG, Gazadar AF, Minna JD, Mitchell JB; Evaluation of a tetrazolium-based semiautomated colormeteric assay: assessment of c he mo sensitivity testing. Cancer Research, 1987, 47: 943-946.

我们使用人肿瘤细胞系进行细胞毒性分析。在含有不同化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8 的正常培养基中培养 31 种人肿瘤细胞株，人肿瘤细胞系购自 ATCC和 NCI，用含 10 % FBS 的 DMEM培养液，在含 5%C02 的 370C孵箱中培养。用胰蛋白酶消化汇合的细胞，经培养液洗涤后计数。向 96孔培养板的每孔中加入 3000〜6000个细胞，孵育 16小时或 24小时。然后向孔内加入不同浓度的化合物 zhangnan-5 ^ zhangnan-7 ^ 和 zhangnan-8 o 继续培养 72小时后，对药物处理组细胞和对照组细胞进行 MTT试验，测定细胞存活情况。结果如表 1所示，表 1显示化合物 _Zh_angn_an-5、 zhangnan-7, 和 zhangnan-8广泛抑制人体肿瘤细胞株，大多数人体肿瘤细胞尤其对化合物 zhangnaii-5敏感。有些肿瘤细胞对这种化合物的 EC50低于 Ο.ΐ μ m。但是，正常人乳腺上皮细胞（MCF— 10a ) 和正常小鼠成纤维细胞（MEF)对化合物 zhangnan-5、 zhangnan-7,和 zhangnan-8 不敏感，即便化合物的浓度高达 30 μ m，这些细胞仍然生长良好。表 1 化合物 zhangnan-5 zhangnan-7 和 zhangnan-8 We used human tumor cell lines for cytotoxicity analysis. 31 human tumor cell lines were cultured in normal medium containing different compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8. Human tumor cell lines were purchased from ATCC and NCI, and cultured in DMEM containing 10% FBS. Cultured in a 370C incubator containing 5% CO 2 . Confluent cells were digested with trypsin, washed after washing with culture medium and counted. 3000 to 6000 cells were added to each well of a 96-well culture plate, and incubated for 16 hours or 24 hours. Then, different concentrations of the compounds zhangnan-5 ^ zhangnan-7 ^ and zhangnan-8 o were added to the wells for 72 hours. After the cells were treated with the MTT test, the cell survival was measured. The results are shown in Table 1. Table 1 shows that the compounds _Z h _an gn _an -5, zhangnan-7, and zhangnan-8 broadly inhibit human tumor cell lines, and most human tumor cells are particularly sensitive to the compound zhangnaii-5. Some tumor cells have an EC50 of less than Ο.ΐ μ m for this compound. However, normal human mammary epithelial cells (MCF-10a) and normal mouse fibroblasts (MEF) are not sensitive to the compounds zhangnan-5, zhangnan-7, and zhangnan-8, even if the concentration of the compound is as high as 30 μm, these cells Still growing well. Table 1 Compound zhangnan-5 zhangnan-7 and zhangnan-8

体外抑制人肿瘤细胞生长细胞类型 zhangnan-5 zhangnan-7 zhangnan-8 人肿瘤细胞株 EC50 ( uM ) EC50 ( uM ) EC50 ( uM )Inhibition of human tumor cell growth in vitro Cell type zhangnan-5 zhangnan-7 zhangnan-8 human tumor cell line EC50 ( uM ) EC50 ( uM ) EC50 ( uM )

LnCAP *** 0.69 15.6 7.8 LnCAP prostate 0.69 15.6 7.8

D145 *** 0.85 14.8 8.4 D145 prostate 0.85 14.8 8.4

PC3 *** 0.91 16.2 8.9 PC3 prostate 0.91 16.2 8.9

HCT-116 结肠 0.09 15.1 7.8 HCT-116 colon 0.09 15.1 7.8

Widr 结肠 0.28 14.8 8.1 Widr colon 0.28 14.8 8.1

HT29 结肠 0.47 13.9 8.9 HT29 colon 0.47 13.9 8.9

LoVo 结肠 0.15 15.2 9.6 LoVo colon 0.15 15.2 9.6

CCL-225 结肠 0.23 16.1 8.6 CCL-225 colon 0.23 16.1 8.6

CCL-247 结肠 0.52 15.6 8.2 CCL-247 colon 0.52 15.6 8.2

NCI-H23 肺 0.32 12.8 6.7 NCI-H23 lung 0.32 12.8 6.7

A549 肺 0.54 13.1 6.9 A549 Lungs 0.54 13.1 6.9

MDA-MB-231 乳腺 0.47 17.5 7.2 MDA-MB-231 Mammary 0.47 17.5 7.2

MDA-MB-435 乳腺 0.55 19.3 7.9 MDA-MB-435 Mammary gland 0.55 19.3 7.9

AU-565 乳腺 0.59 18.2 6.8 AU-565 Mammary 0.59 18.2 6.8

BT-549 乳腺 0.67 17. 4 7.4 BT-549 Breast 0.67 17. 4 7.4

MCF-7 乳腺 0.58 16.9 7.8 MCF-7 breast 0.58 16.9 7.8

Caki-1 肾 0.98 20.1 9.2 Caki-1 kidney 0.98 20.1 9.2

ACHN 肾 1.2 18.3 8.9 ACHN kidney 1.2 18.3 8.9

786-0 肾 3.1 16.8 8.1 786-0 kidney 3.1 16.8 8.1

SN12C 肾 1.3 17.5 7.6 SN12C Kidney 1.3 17.5 7.6

SKOV3 卵巢 1.5 16.9 6.9 SKOV3 ovary 1.5 16.9 6.9

IGROV1 卵巢 2.1 17.4 7.2 IGROV1 Ovary 2.1 17.4 7.2

Mid PaCa-2 胰腺 0.93 18.9 9.4 Mid PaCa-2 Pancreas 0.93 18.9 9.4

U-251 恶性胶质瘤 0.089 17.8 5.6 U-251 Malignant glioma 0.089 17.8 5.6

SK-MEL-5 皮肤（黑色 0.093 18.5 4.9 SK-MEL-5 skin (black 0.093 18.5 4.9

G-361 皮肤（黑色 0.087 19. 3 5.3 G-361 skin (black 0.087 19. 3 5.3

正常乳腺上 >30 Normal breast >30

MCF-lOa >30 >30 MCF-lOa >30 >30

皮细胞 Skin cell

SGC-7901 胃 0.89 18.5 8.9 SGC-7901 stomach 0.89 18.5 8.9

EC 109 食管 0.78 17.9 9.5 EC 109 esophagus 0.78 17.9 9.5

CNE-2Z 鼻咽 1.3 19.2 10.2 CNE-2Z Nasopharyngeal 1.3 19.2 10.2

Raji 淋巴瘤 0.098 20.5 6.9 Raji lymphoma 0.098 20.5 6.9

Jurkat 白血病 T细 0.14 21.3 7.1 Jurkat leukemia T fine 0.14 21.3 7.1

正常小鼠成 Normal mouse

MEF >30 >30 >30 MEF >30 >30 >30

纤维细胞 Fibroblast

化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8或其各种形式形成的盐可作为抗肿瘤药物的原料药，该形成的盐是由衍生物与碱反应生成的，碱可采用各种形式的碱，可以是无机碱如氢氧化钠、氢氧化钾等，也可以是有机碱如碱金属醇盐等，化合物 _Zh_ang_nan-5、 zhangnan-7,和 zhangnan-8或其各种形式形成的盐可与药用载体和 / 或赋形剂制成口服制剂或注射剂制的抗肿瘤药物，药用载体和 /或赋形剂包括谷物油，羧甲基纤维素钠等。 Compound zhangnan-5 ^ zhangnan-7^ and zhangnan-8 or its various forms The formed salt can be used as a raw material for antitumor drugs, and the formed salt is formed by reacting a derivative with a base, and the base can be used in various forms of a base, and may be an inorganic base such as sodium hydroxide or potassium hydroxide. It may be an organic base such as an alkali metal alkoxide, compound _{_{_{Z h an g nan -5, zhangnan}}} -7, or a salt thereof and various forms zhangnan-8 may be formed with pharmaceutically acceptable carriers and / or excipients formulated for oral The antitumor drug prepared by the preparation or the injection, the pharmaceutically acceptable carrier and/or the excipient includes cereal oil, sodium carboxymethylcellulose, and the like.

(2) 化合物 zhangnan-5、 zhangnan-7^ 和 zhangnan-8对 C8166 和 PBMC细胞的毒性： (2) Toxicity of compounds zhangnan-5, zhangnan-7^ and zhangnan-8 to C8166 and PBMC cells:

取对数生长期的 C8166 (人 T淋巴细胞系）和 PBM (用淋巴细胞分离液自正常人外周血分离，贴壁的单个核细胞从 PBMC中分离）细胞，离心（1 000 r，min-l， 10 min)后将细胞重悬，浓度分别调至 4 X105 / ml和 2X106 / ml。在 96孔细胞培养板上将被检样品 2倍倍比稀释，共设 6个梯度，每个梯度设 3个重复孔，另设阳性对照组及细胞对照组。分别加细胞悬液后，置 370C、 5%C02培养箱中孵育。第 7天用 MTT法测定样品对细胞的毒性。按下列公式计算被检样品对细胞生长的抑制率，并计算 IC50 (50 %inhibition concentration) 值。细胞生长抑制率（％ ) = (1- 实验孔 OD值 I对照孔（OD值） X100% Logarithmic growth phase C8166 (human T lymphocyte lineage) and PBM (separated from normal human peripheral blood with lymphocyte separation solution, adherent mononuclear cells were isolated from PBMC) cells, centrifuged (1 000 r, min- After l, 10 min), the cells were resuspended and the concentrations were adjusted to 4 X105 / ml and 2X106 / ml, respectively. The sample to be tested was diluted 2 times in a 96-well cell culture plate, and a total of 6 gradients were set, and 3 replicate wells were set for each gradient, and a positive control group and a cell control group were additionally set. After adding the cell suspension separately, incubate in a 370C, 5% CO 2 incubator. On day 7, the toxicity of the sample to the cells was determined by the MTT method. The inhibition rate of the test sample on cell growth was calculated according to the following formula, and the IC50 (50% inhibition concentration) value was calculated. Cell growth inhibition rate (%) = (1 - experimental well OD value I control well (OD value) X100%

23-轻基白桦酸、化合物 zhangnan-5^ zhangnan-7^ 和 zhangnan-8 对 C8166细胞的 IC50值分另 [J为 15.1±3.57uM、 2.3±0.47uM、 25 ±5.2uM和 10.3±2.9uM，而阳性对照 AZT在 3.437mM时对 50% C8166 细胞产生毒性。 23-羟基白桦酸、化合物 zhangnan-5、 zhangnaii-7、和 zhangnaii-8对正常人 PBMC的 IC50值分另 [J为 14.9 ±3.46 uM、 2.4±0.38uM、 25.8±4.9uM和 11.6±3.5uM。阳性对照药物 SCH— C (治疗艾滋病的抗逆转录病毒药物，是 CCR 的拮抗剂，只能阻止 R 型病毒与细胞结合，对 X 型病毒无效。分子量 (MW)为 557。将 184. 1 m 的 SCH_C溶解到 3. 3 ml DMSO中，得到 lOOummol 的贮存液，于 4°C或一 20°C保存。）对 PBMC 的 IC50为 82. 667ummol。 23-light-based betulinic acid, compound zhangnan-5^ zhangnan-7^ and zhangnan-8 were equally divided into IC50 values of C8166 cells [J was 15.1±3.57uM, 2.3±0.47uM, 25±5.2uM and 10.3±2.9uM The positive control AZT was toxic to 50% C8166 cells at 3.437 mM. The IC50 values of 23-hydroxy-cedaric acid, compound zhangnan-5, zhangnaii-7, and zhangnaii-8 for normal human PBMC were separately [J was 14.9 ± 3.46 uM, 2.4 ± 0.38 uM, 25.8 ± 4.9 uM, and 11.6 ± 3.5 uM. . The positive control drug SCH-C (anti-retroviral drug for the treatment of AIDS, is an antagonist of CCR, can only prevent the R-type virus from binding to cells, and is ineffective against type X virus. The molecular weight (MW) is 557. Will be 18.1 m The SCH_C was dissolved in 3.3 ml of DMSO to obtain a stock solution of lOOummol, which was stored at 4 ° C or 20 ° C.) for PBMC The IC50 is 82. 667 ummol.

( 3 ) 化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8对病毒 HIV-1IIIB诱导 C8166细胞形成合胞体的抑制作用：（可参考文献姜海鸥;汪旭，李汝润，陈大刚，贲昆龙. 复方三黄散胶囊体外抗艾滋病病毒的实验研究， [J】. 山东中医杂志，2005.8(1):21-26. ) (3) The inhibitory effects of the compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 on the formation of syncytia in C8166 cells induced by HIV-1IIIB: (Refer to the literature Jiang Haiou; Wang Xu, Li Yirun, Chen Dagang, Qu Kunlong. Compound Sanhuang Experimental study on anti-HIV in vitro of scattered capsules, [J]. Shandong Journal of Traditional Chinese Medicine, 2005.8(1): 21-26.

在 96孔平底培养板上，将待测物用培养基进行 2倍稀释，共 8 个稀释度，每孔 lOOul设 2个重复孔，同时设置阳性对照组、细胞对照组和病毒对照组。每孔加 4 X 105 / ml的 C8166细胞 8 0 ul , 然后每孔加入 20ul 病毒 HIV-1IIIB 上清，每孔的终末体积为 200 ul，置 37 °C ， 5%C02 培养箱内培养。感染后第 3 天观察化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8对合胞体形成的影响。在倒置显微镜下，计数病毒 HIV-1IIIB诱导 C8166细胞形成的合胞体数。 EC50 为抑制合胞体形成达 50%时的化合物 zhangnan-5、 zhangnan-7, 和 zhangnan-8的浓度。以下列公式计算样品对合胞体形成的抑制率：合胞体形成的抑制率（％) = (1 - 实验孔合胞体数 I对照孔合胞体数) On a 96-well flat-bottomed culture plate, the test substance was diluted twice with a medium for 8 dilutions, and 2 replicate wells per well were set, and a positive control group, a cell control group, and a virus control group were set at the same time. Add 4 X 105 / ml of C8166 cells per well to 80 μl, then add 20 ul of virus HIV-1IIIB supernatant to each well. The final volume of each well is 200 ul, and incubate at 37 °C in a 5% CO 2 incubator. The effects of the compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 on syncytia formation were observed on the third day after infection. Under an inverted microscope, the virus HIV-1IIIB was counted to induce the number of syncytia formed by C8166 cells. EC50 is the concentration of the compounds zhangnan-5, zhangnan-7, and zhangnan-8 when the syncytium formation is inhibited by 50%. The inhibition rate of the syncytium formation by the sample was calculated by the following formula: Inhibition rate of syncytium formation (%) = (1 - number of syncytium cells in the experiment I number of syncytium cells in the control well)

X 100% 阳性对照 AZT显示出有效的抑制 HIV— 1诱导 C8166细胞形成合胞体，其 EC50为 1.39uM，治疗指数 TI值为 2473。 23-羟基白桦酸、化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8的 EC50 值分另 (J为 1.2 ± 0.23uM、 0.5 ± 0.07uM 、 0.0019 ± 0.0002uM、和 1.1 ± 0.09uM ; 其治疗指数分别为 13、 4.6、 13158、 9.4。化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8对 HIV-1IIIB诱导合胞体形成的抑制作用随其浓度的增加而增强，与阳性对照药物 AZT 相比， zhangnaii-7 治疗指数（TI)最高，对合胞体的形成显示出明显的抑制作用。 X 100% positive control AZT showed potent inhibition of HIV-1 induced C8166 cell formation of syncytia with an EC50 of 1.39 uM and a therapeutic index TI of 2473. The EC50 values of 23-hydroxy-cedaric acid, compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 were further (J was 1.2 ± 0.23 uM, 0.5 ± 0.07 uM, 0.0019 ± 0.0002 uM, and 1.1 ± 0.09 uM; The indices were 13, 4.6, 13158, and 9.4. The inhibitory effects of the compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 on HIV-1IIIB-induced syncytia formation increased with increasing concentration, compared with the positive control drug AZT. The zhangnaii-7 treatment index (TI) was the highest, showing a significant inhibitory effect on the formation of syncytia.

( 4 )定量酶联免疫吸附法（EIISA ) 检测化合物对病毒 HIV-lAda-M p24表达的抑制作用：（可参考文献姜海鸥；汪旭，李汝润，陈大刚，贲昆龙. 复方三黄散胶囊体外抗艾滋病病毒的实验研究， [J】. 山东中医杂志，2005.8(1):21-26. ) 在 96孔培养板上，用多头加样器对样品进行 3倍倍比稀释，设 6个稀释度，每个稀释度设三个重复孔，每孔 100ul。同时设阳性对照组 SCH— C (治疗艾滋病的抗逆转录病毒药物，是 CCR 的拮抗剂，只能阻止 R 型病毒与细胞结合，对 X 型病毒无效。分子量 (MW)为 557。将 184. 1 m 的 SCH— C溶解到 3 . 3 ml DMSO中，得到 lOOummol 的贮存液，于 4 °C或一 20 °C保存。；)、病毒对照组和细胞 +病毒对照组。在 96 孔平底培养板上每孔分别加入含有 1000〜5000 pg / well P24 抗原的病毒贮存液 25 ul，充分混匀，加入单个核细胞 PBMC悬液 75 ul (3 X 105 细胞数 /孔），置 37°C， 5%C02培养箱中培养到第 3天时每孔补加 100 ul培养基，第 7天时收集细胞培养上清供 p24抗原测定，按 p24试剂盒说明用 EILSA法检测各样品细胞培养孔上清的病毒 HIV-l p24抗原浓度，以下列公式计算样品对病毒的抑制率：样品对病毒 p24抗原的抑制率（％) = (1 - 实验孔 OD值 /细胞加病毒对照孔 OD值） X 100% ，以 Reed and Mueneh法计算出被测样品的 EC50 , 并进一步计算出治疗指数（TI) (TI = IC50 I EC50)。 (4) Quantitative enzyme-linked immunosorbent assay (EIISA) to detect compounds against the virus HIV-lAda-M Inhibition of p24 expression: (Refer to the literature Jiang Haiou; Wang Xu, Li Yurun, Chen Dagang, Qu Kunlong. Experimental study on anti-HIV virus in vitro by Compound Sanhuang Powder Capsule, [J]. Shandong Journal of Traditional Chinese Medicine, 2005.8(1): 21- 26.) On a 96-well culture plate, the sample was diluted 3 times with a multi-headed sampler, and 6 dilutions were set. Three replicate wells were set for each dilution, 100 ul per well. At the same time, the positive control group SCH-C (anti-retroviral drug for the treatment of AIDS, is an antagonist of CCR, can only prevent the R virus from binding to the cell, and is ineffective against the X virus. The molecular weight (MW) is 557. Will be 184. 1 m of SCH-C was dissolved in 3.3 ml of DMSO to obtain a stock solution of lOOummol, stored at 4 ° C or 20 ° C.;), virus control group and cell + virus control group. Add 25 ul of virus stock solution containing 1000~5000 pg / well P24 antigen to each well of 96-well flat-bottomed culture plate, mix well, add 75 ul of single-core PBMC suspension (3 X 105 cells/well). At 37 ° C, 100 ul of medium was added to each well on the third day of culture in 5% CO 2 incubator. The cell culture supernatant was collected for p24 antigen determination on the 7th day, and each sample cell was detected by EILSA method according to the p24 kit. The concentration of the virus HIV-l p24 antigen in the supernatant of the well was calculated, and the inhibition rate of the virus by the sample was calculated by the following formula: The inhibition rate of the sample against the virus p24 antigen (%) = (1 - OD value of the test well / OD value of the cell plus virus control well) X 100%, calculate the EC50 of the sample by Reed and Mueneh method, and further calculate the therapeutic index (TI) (TI = IC50 I EC50).

当以试剂盒测定 HIVp24抗原的含量时，发现加入的病毒量 (V)为 896. 3 pg / ml , 而病毒增值对照（病毒加细胞， V+ C) 的病毒 p24 含量为 1 452. 9 pg / ml o 阳性对照 SCH_C、 23-羟基白桦酸、化合物 zhangnan-5、 zhangnan-7 ^ 和 zhangnan-8在所测定的范围内都达到了 50%以上的抑制率，阳性对照 SCH— C抑制 HIV-lAda-M p24抗原表达，其 EC50为 0.032 ummol , 治疗指数为 2583。 23- 羟基白桦酸、化合物 zhangnan-5、zhangnan-7、和 zhangnan-8 EC50 值分另 (J为 1.9 ± 0.39uM、 0.7 ± 0.19uM 、 0.0026 ± 0.0005uM、和 1.6 ± 0.23uM，其治疗指数分别为 7.8、 3.4、 9923和 7.3。 23-羟基白桦酸、化合物 zhangnan-5、 zhangnan-7^ 和 zhangnan-8都具有对 HIV-lAda-M的抑制作用，其中化合物 zhangnan-7对 HIV-lAda-M 的抑制活性最强，并且明显强于阳性对照化合物 SCH_ C，而且对 HIV-lAda-M的抑制作用与其浓度存在着明显的量效关系。 When the amount of the virus (V) was found to be 896. 3 pg / ml, and the viral p24 content of the virus-increasing control (virus plus cells, V + C) was 1 452. 9 pg / The ml o positive control SCH_C, 23-hydroxy-cedaric acid, the compounds zhangnan-5, zhangnan-7 ^ and zhangnan-8 all achieved an inhibition rate of more than 50% in the range measured, and the positive control SCH-C inhibited HIV-lAda. -M p24 antigen expression with an EC50 of 0.032 ummol and a therapeutic index of 2583. 23-hydroxy-cedaric acid, compound zhangnan-5, zhangnan-7, and zhangnan-8 EC50 values were further (J 1.9 ± 0.39uM, 0.7 ± 0.19uM, 0.0026 ± 0.0005uM, and 1.6 ± 0.23uM, the therapeutic index They are 7.8, 3.4, 9923 and 7.3. 23-hydroxy betulinic acid, compound zhangnan-5, zhangnan-7^ and zhangnan-8 all have The inhibitory effect of HIV-lAda-M, in which the compound zhangnan-7 has the strongest inhibitory activity against HIV-1Ada-M, and is significantly stronger than the positive control compound SCH_C, and the inhibitory effect on HIV-1Ada-M and its concentration exist. Obvious dose-effect relationship.

化合物 zhangnan-5 ^ zhangnan-7^ 和 zhangnan-8或其各种形式形成的盐可作为抗艾滋病药物的原料药，该形成的盐是由衍生物与碱反应生成的，碱可采用各种形式的碱，可以是无机碱如氢氧化钠、氢氧化钾等，也可以是有机碱如碱金属醇盐等，化合物 _Zh_angn_an-5、 zhangnan-7,和 zhangnan-8或其各种形式形成的盐可与药用载体和 / 或赋形剂制成口服制剂或注射剂制的抗艾滋病药物，药用载体和 /或赋形剂包括谷物油，羧甲基纤维素钠等。 The salts formed by the compounds zhangnan-5 ^ zhangnan-7^ and zhangnan-8 or various forms thereof can be used as a raw material for anti-AIDS drugs, and the formed salts are formed by reacting a derivative with a base, and the base can be used in various forms. The base may be an inorganic base such as sodium hydroxide, potassium hydroxide or the like, or an organic base such as an alkali metal alkoxide or the like, a compound _Z h _an gn _an -5, zhangnan-7, and zhangnan-8 or various thereof. The formally formed salt may be formulated into an oral preparation or an injection-preventing anti-AIDS drug with a pharmaceutically acceptable carrier and/or excipient, and the pharmaceutically acceptable carrier and/or excipient includes cereal oil, sodium carboxymethylcellulose, and the like.

( 5 )化合物 zhangnaii-5在小鼠体内的药代动力学研究：（可参考文献 Singh SS, Shah H, Gupta S, Jain M, Sharma K, Thakkar P, Shah R. Liquid chromatography— electrospray ionisation mass spectrometry method for the determination of escitalopram in human plasma and its application in bioequivalence study. J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Nov 25;811(2):209-15. ) (5) Pharmacokinetic study of the compound zhangnaii-5 in mice: (Refer to Singh SS, Shah H, Gupta S, Jain M, Sharma K, Thakkar P, Shah R. Liquid chromatography - electrospray ionisation mass spectrometry Method for the determination of escitalopram in human plasma and its application in bioequivalence study. J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Nov 25;811(2):209-15.

取 4只 8 周龄 BlebC 小鼠（2 只雄性和 2 只雌性），单次剂量 100mg/kg给予化合物 zhangnan-5。分别于口饲给药后 0.5、 1、 3、 6、 12、 24、 48、 72 小时，经眼静脉取血并制得血浆，以测定给予化合物 zhangnan-5的血浆浓度。表 2. 化合物 zhangnan-5在小鼠体内的药代动力学参数 Four 8-week-old BlebC mice (2 males and 2 females) were given a single dose of 100 mg/kg of compound zhangnan-5. Plasma was taken from the ocular vein at 0.5, 1, 3, 6, 12, 24, 48, and 72 hours after oral administration, to determine the plasma concentration of the compound zhangnan-5. Table 2. Pharmacokinetic parameters of compound zhangnan-5 in mice

( 6 ) 给药后化合物 zhangnaii-5对小鼠的急性毒性研究：（可参考文献 Gol'dberg LE, Stepanova ES, Vertogradova TP, Shevniuk LA, Shepelevtseva G. Preclinical toxicological study of the new antibiotic eremomycin. Its acute toxicity for laboratory animals, Antibiot Med Biotekhnol. 1987, 32(12): 910-5. )

(6) Acute toxicity study of compound zhangnaii-5 on mice after administration: (Refer to Gol'dberg LE, Stepanova ES, Vertogradova TP, Shevniuk LA, Shepelevtseva G. Preclinical toxicological study of the new antibiotic eremomycin. Its acute Toxicity for laboratory animals, Antibiot Med Biotekhnol. 1987, 32(12): 910-5. )

取两组 8周龄的 BlabC小鼠，每组 10只（5只雄性和 5只雌性），分别以单次剂量 500mg/kg和多次剂量 200mg/kg ( QDX15 ) 给予化合物 _Zh_angn_an-5 (用玉米油或载体配制），然后分别观察 1周和 4周。每两天称体重。试验结束后，将受试小鼠处死，进行病理分析。结果显示，以单次剂量 500mg/Kg和多次剂量 200mg/kg ( QDX15 ) 给予化合物 _Zh_{a n}gn_an-5，均未观察到毒性，所有受试小鼠生长良好，无一死亡。因此给予化合物 zhangnan-5是具有很大开发前景的广谱抗肿瘤新药，化合物 zhangnaii-5可作为抗肿瘤药物的原料药，优先作为抗恶性实体瘤药物的原料药，化合物 zhangnaii-5 也可与药用载体和 / 或赋形剂制成抗肿瘤药物。所述药用载体和 /或赋形剂例如谷物油，羧甲基纤维素钠等。化合物 zhangnan-5 —般口服推荐剂量为每天 165mg/体表面积 m2，连续三周，休息一周为一疗程。化合物 zhangnaii-5每天总剂量一次于早餐后半小时口服，具体病例可由医师根据病情调整。 Take two BlabC 8 weeks old mice, 10 per group (5 males and 5 females), respectively, in a single dose of 500mg / kg and multiple doses of 200mg / kg (QDX15) to give compound _Z h _{_an} gn _an -5 (prepared with corn oil or vehicle) and observed for 1 week and 4 weeks, respectively. Weigh the weight every two days. After the end of the experiment, the test mice were sacrificed for pathological analysis. The results showed that the compound _Z h _an gn _an -5 was administered in a single dose of 500 mg/kg and multiple doses of 200 mg/kg (QDX15), and no toxicity was observed. All the mice grew well without death. Therefore, the compound zhangnan-5 is a broad-spectrum anti-tumor drug with great development prospects. The compound zhangnaii-5 can be used as a drug substance for anti-tumor drugs, and is preferred as a drug substance for anti-malignant solid tumor drugs. Compound zhangnaii-5 can also be used. Pharmaceutically acceptable carriers and/or excipients are used to make antitumor drugs. The pharmaceutically acceptable carrier and/or excipients are, for example, cereal oil, sodium carboxymethylcellulose, and the like. The general recommended dose of the compound zhangnan-5 is 165 mg/body surface area m2 per day for three consecutive weeks, and one week of rest is a course of treatment. The total daily dose of the compound zhangnaii-5 is orally administered half an hour after breakfast, and the specific case can be adjusted by the physician according to the condition.

( 7 ) 人肿瘤异种移植模型和裸小鼠肿瘤移植模型的给药： (7) Administration of human tumor xenograft models and nude mouse tumor transplantation models:

(可参考文献 Harrison SD Jr； Growth of human tumor cells in athymic mice; J Natl Cancer Inst. 1991 Oct 16 ;83(20): 1509. ) (References Harrison SD Jr; Growth of human tumor cells in athymic mice; J Natl Cancer Inst. 1991 Oct 16 ; 83(20): 1509. )

T细胞缺陷裸小鼠（nu/nu )，雄性， 6周龄，购自 Charles River 实验室，按照大学动物饲养和使用委员会指南饲养于无病原体环境。将 5 X 106个 LNCaP、 NCI-H23 , HCT-116、 G-361、 U-251细胞分别悬浮于 0.2mlHBSS 或基质胶（ 50_: 50，v/v ) 中，皮下接种到小鼠的侧腹部区域。当肿瘤平均直径达 7〜8mm 时，选出肿瘤大小为 100〜 200mm3 的小鼠，分为给予玉米油配制的化合物 zhangnan-5 的处理组和只给予空载体（玉米油）的对照组。为保证化合物 zhangnan-5 处理组和对照组在处理开始时肿瘤体积分布大致相等，将小鼠分成三类：肿瘤体积小（长度 <4mm )，肿瘤体积中等（4〜8mm )，肿瘤体积大（>8mm )。对照组，化合物 zhangnan-5处理组中来自相同类别的小鼠数目大致相等。在口服给药试验中，将化合物 zhangnan-5 (30mg/kg)溶解于玉米油中。每日管饲给予单次剂量 200μ1药液（用玉米油配制，含 0.75mg 化合物 zhangnan-5 ) ，每周 5次，持续 3周；对照组按同样方法给予安慰剂（玉米油）。小鼠单独饲养，允许自由采食。抗肿瘤效应评价：每 3〜4天沿两个垂直方向对肿瘤进行测量。肿瘤体积根据以下公式计算： T cell deficient nude mice (nu/nu), male, 6 weeks old, purchased from Charles River Laboratories, were housed in a pathogen free environment according to the guidelines of the University Animal Feeding and Use Committee. The 5 X 106 th LNCaP, NCI-H23, HCT- 116, G-361, U-251 cells were suspended in Matrigel or 0.2mlHBSS _{(50: 50, v / v} ) , the mice were subcutaneously inoculated into abdominal side region. When the average diameter of the tumor reaches 7~8mm, the tumor size is selected to be 100~ The mice of 200 mm3 were divided into a treatment group of the compound zhangnan-5 formulated with corn oil and a control group given only the empty vector (corn oil). To ensure that the compound volume of the zhangnan-5 treatment group and the control group were approximately equal at the beginning of the treatment, the mice were divided into three categories: small tumor volume (length < 4 mm), medium tumor volume (4 to 8 mm), and large tumor volume ( >8mm). In the control group, the number of mice from the same category in the compound zhangnan-5 treatment group was approximately equal. In an oral administration test, the compound zhangnan-5 (30 mg/kg) was dissolved in corn oil. Daily gavage was given a single dose of 200 μl (prepared with corn oil containing 0.75 mg of compound zhangnan-5) 5 times a week for 3 weeks; the control group was given a placebo (corn oil) in the same manner. Mice were housed individually, allowing free access to food. Evaluation of anti-tumor effect: Tumors were measured in two perpendicular directions every 3 to 4 days. The tumor volume is calculated according to the following formula:

V= (a2 X b)/2 其中 a是肿瘤宽度（较小直径）， b是长度（较大直径）。每个肿瘤的相对肿瘤体积（RTV )定义为给定时间点的肿瘤体积与处理开始时肿瘤体积的比值。每个处理组均计算平均值。根据以下公式计算肿瘤生长抑制值（TGI ) , 判断抗肿瘤活性： V = (a2 X b)/2 where a is the tumor width (smaller diameter) and b is the length (larger diameter). The relative tumor volume (RTV) of each tumor is defined as the ratio of the tumor volume at a given time point to the tumor volume at the beginning of the treatment. The average was calculated for each treatment group. The tumor growth inhibition value (TGI) was calculated according to the following formula to determine the antitumor activity:

TGI(%)=T/C X 100 其中 T是处理组试验终点（4周） RTV的平均值， C是对照组试验终点 RTV的平均值。采用美国国立癌症研究所的最小抗肿瘤活性标准（T/C 42% )。在化合物 zhangnaii-5 有效抑制多种肿瘤细胞体外增殖的基础上，应用人肿瘤移植裸鼠（皮下接种）评价化合物 zhangnan-5的抗肿瘤作用，受试的肿瘤细胞包括人***癌细胞 LnCAP , 人肺癌细胞 NCI-H23,人结肠癌细胞 HCT-116, 人黑色素瘤细胞 G-361和人恶性胶质瘤细胞 U-251。结果如图 1一5所示，化合物 zhangnaii-5强烈抑制这三种肿瘤生长，其 TGI值分别为 5%， 0% , 6.9% , 10.8% , 和 12.5%。同时未发现化合物 zhangnan-5具有明显的副作用，包括体重的变化。这些数据强烈提示，化合物 zhangnan-5是开发新的抗肿瘤药物的一个很好的候选化合物。 TGI (%) = T / CX 100 where T is the mean of the RTV of the treatment group end point (4 weeks) and C is the mean of the RTV of the control group end point. The National Cancer Institute's minimum anti-tumor activity standard (T/C 42%) was used. On the basis of the compound zhangnaii-5 effectively inhibiting the proliferation of various tumor cells in vitro, human tumor-transplanted nude mice (subcutaneous vaccination) were used to evaluate the anti-tumor effect of the compound zhangnan-5, and the tumor cells tested included human prostate cancer cells LnCAP, human Lung cancer Cell NCI-H23, human colon cancer cell HCT-116, human melanoma cell G-361 and human malignant glioma cell U-251. The results are shown in Figure 1-5. Compound zhangnaii-5 strongly inhibited the growth of these three tumors with TGI values of 5%, 0%, 6.9%, 10.8%, and 12.5%, respectively. At the same time, the compound zhangnan-5 was not found to have significant side effects, including changes in body weight. These data strongly suggest that the compound zhangnan-5 is a good candidate for the development of new anti-tumor drugs.

Claims

权利要求： Rights request:

1、一种 23-羟基白桦酸衍生物，该衍生物的化学名称为 3-羧基 -23- ¾基白桦酸，化学式为 A 23-hydroxy betulinic acid derivative having the chemical name 3-carboxy-23- 3⁄4-based betulinic acid, the chemical formula

2、制备权利要求 1所述的 23-羟基白桦酸衍生物的方法，包括以下步骤： 2. A method of preparing a 23-hydroxy betulinic acid derivative according to claim 1, comprising the steps of:

( 1 )将 23-羟基白桦酸溶解于吡啶中，搅拌均匀，缓慢滴加醋酸酑，搅拌进行反应，然后缓慢滴加氯化铵饱和溶液，直至无气泡产生，乙酸乙酯萃取后合并有机层，饱和氯化钠水溶液洗有机层，再用无水硫酸钠干燥，旋干有机溶剂乙酸乙酯后上柱分离，除回收部分原料外，分别得到化合物 3， 23-二乙酰基 -23-羟基白桦酸， 23-二乙酰基 -23- 羟基白桦酸和 3-二乙酰基 -23-羟基白桦酸； (1) Dissolve 23-hydroxy betulinic acid in pyridine, stir evenly, slowly add cesium acetate, stir to carry out the reaction, then slowly add a saturated solution of ammonium chloride until no bubbles are generated, and extract the organic layer after ethyl acetate extraction. The organic layer was washed with a saturated aqueous solution of sodium chloride and dried over anhydrous sodium sulfate. The organic solvent ethyl acetate was evaporated and then applied to the column, and the fractions were recovered to obtain the compound 3, 23-diacetyl-23-hydroxyl. Birch acid, 23-diacetyl-23-hydroxycetanoic acid and 3-diacetyl-23-hydroxycetanoic acid;

( 2 ) 将 23-二乙酰基 -23-羟基白桦酸溶解分散于二氯甲烷中，于冰浴下加入氧化剂二环己基碳二亚胺后，于室温搅拌进行反应，旋干溶剂二氯甲烷后用乙酸乙酯溶解后用粗硅胶进行柱分离，得到化合物 3-羧基 -23-二乙酰基 -23-羟基白桦酸； (2) Dissolving 23-diacetyl-23-hydroxy betulinic acid in dichloromethane, adding the oxidizing agent dicyclohexylcarbodiimide in an ice bath, stirring at room temperature, and spinning the solvent to dichloromethane. After dissolving with ethyl acetate, the column was separated with crude silica gel to obtain the compound 3-carboxy-23-diacetyl-23-hydroxybutteric acid;

( 3 ) 取 3-羧基 -23-二乙酰基 -23-羟基白桦酸分散溶解于甲醇中，加入碳酸钾的饱和溶液后，于室温搅拌进行反应，乙酸乙酯萃取后合并有机层，饱和氯化钠水溶液洗有机层，再用无水硫酸钠干燥，旋干有机溶剂乙酸乙酯后上柱分离，得化合物 3-羧基 -23-羟基白桦酸。 (3) 3-carboxy-23-diacetyl-23-hydroxy betulinic acid is dispersed and dissolved in methanol. After adding a saturated solution of potassium carbonate, the reaction is stirred at room temperature, and the organic layer is extracted with ethyl acetate. The organic layer was washed with an aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent ethyl acetate was evaporated.

3、一种 23-羟基白桦酸衍生物，该衍生物的化学名称为 3-羧基 -23- 羟基二氢白桦酸，化学式为

3. A 23-hydroxy betulinic acid derivative having the chemical name 3-carboxy-23-hydroxydihydroglycylic acid, the chemical formula

4、制备权利要求 3所述的 23-羟基白桦酸衍生物的方法，包括：取 3-羧基 -23-羟基白桦酸分散溶解于甲醇中，加入还原剂钯碳后密封反应体系，抽真空，充入氢气，并于氢气流中室温搅拌进行反应，在结束反应后滤除钯碳，然后旋干溶剂，得到产物 3-羧基 -23-羟基二氢白桦酸。 4. The method for preparing a 23-hydroxy betulinic acid derivative according to claim 3, comprising: dissolving 3-carboxy-23-hydroxy betulinic acid in methanol, adding a reducing agent palladium carbon, sealing the reaction system, and vacuuming, Hydrogen gas was charged, and the reaction was carried out by stirring at room temperature in a hydrogen stream. After the completion of the reaction, palladium carbon was filtered off, and then the solvent was evaporated to give the product 3-carboxy-23-hydroxydihydroglycolic acid.

5、一种 23-羟基白桦酸衍生物，该衍生物的化学名称为 23-羟基二氢白桦酸，化学式为 5. A 23-hydroxy betulinic acid derivative having the chemical name 23-hydroxydihydrocetanoic acid, the chemical formula

6、制备权利要求 5所述的 23-羟基白桦酸衍生物的方法，包括：取化合物 23-羟基白桦酸分散溶解于甲醇中，加入还原剂钯碳后密封反应体系，抽真空，充入氢气，并于氢气流中室温搅拌进行反应，在结束反应后，滤除钯碳，然后旋干溶剂得到产物 23-羟基二氢白桦酸。 6. A method of preparing a 23-hydroxy betulinic acid derivative according to claim 5, comprising: dissolving a compound 23-hydroxy betulinic acid in methanol, adding a reducing agent palladium carbon, sealing the reaction system, evacuating, and charging hydrogen gas. The reaction was carried out by stirring at room temperature in a hydrogen stream. After the reaction was completed, palladium carbon was filtered off, and then the solvent was evaporated to give the product 23-hydroxydihydroglycolic acid.

7、权利要求 1、 3、 5中任一项所述的 23-羟基白桦酸衍生物或其各种形式形成的盐在制备抗肿瘤药物中的应用。 The use of the 23-hydroxy betulinic acid derivative according to any one of claims 1, 3, or 5 or a salt thereof in various forms for the preparation of an antitumor drug.

8、权利要求 1、 3、 5中任一项所述的 23-羟基白桦酸衍生物或其各种形式形成的盐在制备抗艾滋病药物中的应用。 The use of the 23-hydroxy betulinic acid derivative according to any one of claims 1, 3, or 5 or a salt thereof in various forms for the preparation of an anti-AIDS drug.

9、药物组合物，其中含有权利要求 1、 3、 5中任一项所述的 23- 羟基白桦酸衍生物或其各种形式形成的盐作为有效成分，并含有常规药用载体和 /或赋形剂。 A pharmaceutical composition comprising the 23-hydroxybutteric acid derivative according to any one of claims 1, 3, or 5 or a salt thereof in various forms as an active ingredient, and comprising a conventional Pharmaceutically acceptable carriers and/or excipients.

10、根据权利要求 9所述的药物组合物，该药物组合物制成口服制剂或注射剂，其中所述药用载体和 /或赋形剂包括谷物油，羧甲基纤维素钠。 The pharmaceutical composition according to claim 9, which is formulated into an oral preparation or an injection, wherein the pharmaceutically acceptable carrier and/or excipient comprises a cereal oil, sodium carboxymethylcellulose.